牦牛SMPX基因CDS区克隆及组织表达分析
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摘要
本研究旨在克隆牦牛X染色体相关小肌肉蛋白(small muscle protein X-link,SMPX)基因的CDS区序列,分析该序列所编码蛋白的结构与功能,并检测SMPX基因在牦牛不同组织中的表达情况。运用RT-PCR技术扩增并克隆SMPX基因CDS区序列,分析其氨基酸序列相似性并构建系统进化树;通过在线软件对其理化性质、二级结构和三级结构进行生物信息学分析;采用实时荧光定量PCR方法检测SMPX基因在牦牛右心室、臀大肌、肺脏和大脑4个组织中的表达情况。结果表明,牦牛SMPX基因CDS区全长515 bp,开放阅读框(ORF)长261 bp,编码86个氨基酸。牦牛SMPX氨基酸序列与野牦牛、水牛、家犬、人、白尾鹿德克萨斯亚种、绵羊、黑猩猩、藏羚羊、野猪的相似性分别为100%、97.7%、96.5%、96.5%、95.3%、95.3%、96.5%、94.2%和91.9%,说明其在不同物种间具有较高的保守性。生物信息学分析发现,SMPX蛋白是一种不稳定的亲水性蛋白,二级结构以无规则卷曲和α-螺旋为主,为膜内蛋白,无信号肽和跨膜蛋白;SMPX氨基酸序列共有4个磷酸化位点。亚细胞定位结果表明,SMPX蛋白的分布于细胞核(52.2%)、线粒体(43.5%)和细胞质(4.3%)。实时荧光定量PCR检测结果显示,SMPX基因在牦牛右心室中表达量最高,显著高于其他组织(P<0.05)。本试验结果为深入研究SMPX基因在牦牛中的生理功能和调控机制提供了参考数据。
This study was aimed to clone the CDS sequence of small muscle protein X-link(SMPX) gene,analyze its potential structure and function,and explore the expression levels of SMPX gene in different tissues of Bos grunniens.The CDS sequence of SMPX gene was amplified and cloned by RT-PCR,the amino acid sequence homology of SMPX gene was analyzed,and the phylogenetic tree was constructed.The physical and chemical properties,secondary structure and tertiary structure of SMPX protein were analyzed by bioinformatics softwares.The expression patterns of SMPX gene in right ventricle,gluteus maximus,lung and brain of Bos grunniens were detected by Real-time PCR.The results showed that the length of SMPX gene CDS and ORF sequences were 515 and 261 bp,respectively,which encoded 86 amino acids.The amino acid sequence homology of SMPX gene in Bos grunniens with Bos mutus,Bubalus bubalis,Canis lupus familiaris,Homo sapiens,Odocoileus virginianus texanus,Ovis aries,Pan troglodytes,Pantholops hodgsonii and Sus scrofa were 100%,97.7%,96.5%,96.5%,95.3%,95.3%,96.5%,94.2% and 91.9%,respectively,indicating that it was highly conserved in different species.Bioinformatics analysis represented that SMPX protein was unstable and hydrophilic.The secondary structure of SMPX mainly contained random coils and alpha helix.The SMPX protein had no signal peptide and transmembrane protein, and belonged to intramembrane protein.There were four phosphorylation sites in SMPX protein sequence.The results of subcellular localization showed that SMPX protein was located in the nucleus(52.2%),mitochondria(43.5%) and cytoplasm(4.3%).Real-time PCR results exhibited that the expression level of SMPX gene in right ventricle was significantly higher than gluteus maximus,lung and brain(P<0.05).This study might provide reference data for further study of physiological functions and regulatory mechanisms of SMPX gene in Bos grunniens.
This study was aimed to clone the CDS sequence of small muscle protein X-link(SMPX) gene,analyze its potential structure and function,and explore the expression levels of SMPX gene in different tissues of Bos grunniens.The CDS sequence of SMPX gene was amplified and cloned by RT-PCR,the amino acid sequence homology of SMPX gene was analyzed,and the phylogenetic tree was constructed.The physical and chemical properties,secondary structure and tertiary structure of SMPX protein were analyzed by bioinformatics softwares.The expression patterns of SMPX gene in right ventricle,gluteus maximus,lung and brain of Bos grunniens were detected by Real-time PCR.The results showed that the length of SMPX gene CDS and ORF sequences were 515 and 261 bp,respectively,which encoded 86 amino acids.The amino acid sequence homology of SMPX gene in Bos grunniens with Bos mutus,Bubalus bubalis,Canis lupus familiaris,Homo sapiens,Odocoileus virginianus texanus,Ovis aries,Pan troglodytes,Pantholops hodgsonii and Sus scrofa were 100%,97.7%,96.5%,96.5%,95.3%,95.3%,96.5%,94.2% and 91.9%,respectively,indicating that it was highly conserved in different species.Bioinformatics analysis represented that SMPX protein was unstable and hydrophilic.The secondary structure of SMPX mainly contained random coils and alpha helix.The SMPX protein had no signal peptide and transmembrane protein, and belonged to intramembrane protein.There were four phosphorylation sites in SMPX protein sequence.The results of subcellular localization showed that SMPX protein was located in the nucleus(52.2%),mitochondria(43.5%) and cytoplasm(4.3%).Real-time PCR results exhibited that the expression level of SMPX gene in right ventricle was significantly higher than gluteus maximus,lung and brain(P<0.05).This study might provide reference data for further study of physiological functions and regulatory mechanisms of SMPX gene in Bos grunniens.
引文
[1] 张容昶.中国的牦牛[M].兰州:甘肃科学技术出版社,1989.ZHANG R C.Chinese Yak[M].Lanzhou:Gansu Science & Technology Press,1989.(in Chinese)
[2] 中国畜禽遗传资源状况编委会.中国畜禽遗传资源状况[M].北京:中国农业出版社,2004.EDITORIAL COMMITTEE OF REPORT ON DOMESTIC ANIMAL GENETIC RESOURCES IN CHINA.Report on Domestic Animal Genetic Resources in China[M].Beijing:China Agriculture Press,2004.(in Chinese)
[3] 熊维权.我国牦牛资源开发前景与措施[J].农牧产品开发,1999,1:3.XIONG W Q.Prospects and measures for the development of yak resources in China[J].Development of Agriculture and Animal Husbandry Products,1999,1:3.(in Chinese)
[4] 门小明.肌肉纤维类型组成对猪肉品质的影响及其机理研究[D].无锡:江南大学,2012.MEN X M.Effects and underlying mechanisms of muscle fibre types on pork quality[D].Wuxi:Jiangnan University,2012.(in Chinese)
[5] 邓瑞强.SMPX和STARS基因在大白猪不同生长阶段背腰最长肌中表达差异研究[D].太谷:山西农业大学,2015.DENG R Q.SMPX and STARS genes in different growth stage Large White back longest muscle in the waist expression differences research[D].Taigu:Shanxi Agricultural University,2015.(in Chinese)
[6] PATZAK D,ZHUCHENKO O,LEE C C,et al.Identification,mapping,and genomic structure of a novel X-chromosomal human gene (SMPX) encoding a small muscular protein[J].Human Genetics,1999,105(5):506-512.
[7] PALMER S,GROVES N,SEHINDELER A,et al.The small muscle-specific protein Csl modifies cell shape and promote smyocyte fusion in insulin-like growth factor 1-dependent manner [J].The Journal of Cell Biology,2001,153(5):985-998.
[8] KEMP T J,SADUSKY T J,SIMON M M,et al.Identification of a novel stretch responsive skeletal muscle gene(SMPX) [J].Genomics,2001,72:260-271.
[9] 官红平.猪肌肉生长发育相关基因SMPX、STARS、VGLL2和VGLL4的克隆、定位及其与部分性状的关联分析[D].武汉:华中农业大学,2006.GUAN H P.Cloning,chromosomal localization and association analysis of porcine SMPX,STARS,VGLL2 and VGLL4 genes which relating to muscle development and growth[D].Wuhan:Huazhong Agricultural University,2006.(in Chinese)
[10] EFTEST?L E,ALVER T N,GUNDERSEN K,et al.Overexpression of SMPX in adult skeletal muscle does not change skeletal muscle fiber type or size[J].PLoS One,2014,9(6):e99232.
[11] NIU Z,FENG Y,MEI L,et al.A novel frameshift mutation of SMPX causes a rare form of X-linked nonsyndromic hearing loss in a Chinese family[J].PLoS One,2017,12(5):e0178384.
[12] NIU Z,YAN D,BRESSLER S,et al.A novel splicing mutation in SMPX is linked to nonsyndromic progressive hearing loss[J].International Journal of Pediatric Otorhinolaryngology,2018,104:47-50.
[13] DENG Y,NIU Z,FAN L,et al.A novel mutation in the SMPX gene associated with X-linked nonsyndromic sensorineural hearing loss in a Chinese family[J].Journal of Human Genetics,2018,63(6):723-730.
[14] 施会彬,王玉琴,田志龙,等.伏牛白山羊TACR1基因扩增及生物信息学分析[J].中国畜牧兽医,2018,45(9):2409-2416.SHI H B,WANG Y Q,TIAN Z L,et al.Amplification and bioinformatics analysis of TACR1 gene in Funiu White goat[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2409-2416.(in Chinese)
[15] 罗轶.鸡 FATP1基因cDNA的克隆、组织表达及其生物信息学分析[D].雅安:四川农业大学,2008.LUO Y.cDNA cloning,tissue expression and bioinformatic analysis of fatty acid transport protein 1 in chicken[D].Ya’an:Sichuan Agricultural University,2008.(in Chinese)
[16] 张霞,王淑燕,王配,等.版纳微型猪近交系KITLG基因克隆、组织表达及功能特性分析[J].云南农业大学学报(自然科学),2018,33(2):255-261.ZHANG X,WANG S Y,WANG P,et al.Cloning,tissue expression and functional characteristics of the KITLG gene of Banna Mini-pig inbred line[J].Journal of Yunnan Agricultural University (Natural Science),2018,33(2):255-261.(in Chinese)
[17] 廖珂,柴志欣,钟金城.西藏牦牛HBG基因克隆及生物信息学分析[J].畜牧与兽医,2016,48(3):64-67.LIAO K,CHAI Z X,ZHONG J C.Cloning and bioinformatics analysis on HBG gene in Tibet yak[J].Animal Husbandry & Veterinary Medicine,2016,48(3):64-67.(in Chinese)
[18] 张津闽,张小玉,林森,等.藏系绵羊BMP2基因克隆及组织表达分析[J].基因组学与应用生物学,2018,37(4):1376-1382.ZHANG J M,ZHANG X Y,LIN S,et al.Cloning and tissue expression analysis of BMP2 gene in Tibetan sheep[J].Genomics and Applied Biology,2018,37(4):1376-1382.(in Chinese)
[19] 梁莎莎,庞春英,邓廷贤,等.奶水牛固醇携带蛋白2基因克隆、生物信息学分析及其组织表达检测[J].中国畜牧兽医,2018,45(9):2358-2367.LIANG S S,PANG C Y,DENG T X,et al.Cloning,bioinformatics and tissue expression analysis of SCP2 gene in dairy buffalo[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2358-2367.(in Chinese)
[20] BEATRIZ F,INGRID M P,JUDITH A,et al.The nuclear receptor NOR-1 regulates the small muscle protein,X-linked (SMPX) and myotube differentiation[J].Scientific Reports,2016,6:25944.
[21] LITCHFIELD D W.Protein kinase CK2:Structure,regulation and role in cellular decisions of life and death[J].Biochemical Journal,2003,369(Pt1):1-15.
[22] LARZUL C,LEFAUCHEUR L,ECOLAN P,et al.Phenotypic and genetic parameters for longissimus muscle fiber characteristics in relation to growth,carcass,and meat quality traits in Large White pigs[J].Journal of Animal Science,1997,75:3126-3137.
[23] REN Z Q,XIONG Y Z,DENG C Y,et al.Cloning and identification of porcine SMPX differentially expressed in F1 crossbreds and their parents[J].Acta Biochimica et Biophysica Sinica,2006,38(11):753-758.
[24] BEECKMANN P,SCHR?FFEL J,MOSER G,et al.Linkage and QTL mapping for Sus scrofa chromosome 1[J].Journal of Animal Breeding & Genetics,2015,120(s1):1-10.
[25] DONGIOVANNI P,VALENTI L.Genetics of nonalcoholic fatty liver disease[J].Metabolism Clinical & Experimental,2015,65(8):1026-1037.
[26] 李强子,朱国强,刘吴鑫,等.荷斯坦牛TLR6基因CDS区的生物信息学分析[J].江苏农业学报,2016,32(3):608-614.LI Q Z,ZHU G Q,LIU W X,et al.Bioinformatics analysis of CDS of TLR6 gene in Holstein cattle[J].Jiangsu Journal of Agricultural Sciences,2016,32(3):608-614.(in Chinese)
[27] 营瑞文,张永生,夏欢,等.驴Zfy基因cDNA克隆及生物信息学分析[J].中国畜牧兽医,2018,45(1):39-46.YING R W,ZHANG Y S,XIA H,et al.cDNA cloning and bioinformatics analysis of Zfy gene in Equus asinus[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):39-46.(in Chinese)
[28] SCHINDELER A,LAVULO L,HARVEY R P.Muscle costameric protein,Chisel/Smpx,associates with focal adhesion complexes and modulates cell spreading in vitro via a Rac1/p38 pathway[J].Experimental Cell Research,2005,307(2):367-380.
[29] CHRISTOFFELS V M,HABETS P E,FRANCO D,et al.Chamber formation and morphogenesis in the developing mammalian heart[J].Developmental Biology,2000,223(2):266-278.
[2] 中国畜禽遗传资源状况编委会.中国畜禽遗传资源状况[M].北京:中国农业出版社,2004.EDITORIAL COMMITTEE OF REPORT ON DOMESTIC ANIMAL GENETIC RESOURCES IN CHINA.Report on Domestic Animal Genetic Resources in China[M].Beijing:China Agriculture Press,2004.(in Chinese)
[3] 熊维权.我国牦牛资源开发前景与措施[J].农牧产品开发,1999,1:3.XIONG W Q.Prospects and measures for the development of yak resources in China[J].Development of Agriculture and Animal Husbandry Products,1999,1:3.(in Chinese)
[4] 门小明.肌肉纤维类型组成对猪肉品质的影响及其机理研究[D].无锡:江南大学,2012.MEN X M.Effects and underlying mechanisms of muscle fibre types on pork quality[D].Wuxi:Jiangnan University,2012.(in Chinese)
[5] 邓瑞强.SMPX和STARS基因在大白猪不同生长阶段背腰最长肌中表达差异研究[D].太谷:山西农业大学,2015.DENG R Q.SMPX and STARS genes in different growth stage Large White back longest muscle in the waist expression differences research[D].Taigu:Shanxi Agricultural University,2015.(in Chinese)
[6] PATZAK D,ZHUCHENKO O,LEE C C,et al.Identification,mapping,and genomic structure of a novel X-chromosomal human gene (SMPX) encoding a small muscular protein[J].Human Genetics,1999,105(5):506-512.
[7] PALMER S,GROVES N,SEHINDELER A,et al.The small muscle-specific protein Csl modifies cell shape and promote smyocyte fusion in insulin-like growth factor 1-dependent manner [J].The Journal of Cell Biology,2001,153(5):985-998.
[8] KEMP T J,SADUSKY T J,SIMON M M,et al.Identification of a novel stretch responsive skeletal muscle gene(SMPX) [J].Genomics,2001,72:260-271.
[9] 官红平.猪肌肉生长发育相关基因SMPX、STARS、VGLL2和VGLL4的克隆、定位及其与部分性状的关联分析[D].武汉:华中农业大学,2006.GUAN H P.Cloning,chromosomal localization and association analysis of porcine SMPX,STARS,VGLL2 and VGLL4 genes which relating to muscle development and growth[D].Wuhan:Huazhong Agricultural University,2006.(in Chinese)
[10] EFTEST?L E,ALVER T N,GUNDERSEN K,et al.Overexpression of SMPX in adult skeletal muscle does not change skeletal muscle fiber type or size[J].PLoS One,2014,9(6):e99232.
[11] NIU Z,FENG Y,MEI L,et al.A novel frameshift mutation of SMPX causes a rare form of X-linked nonsyndromic hearing loss in a Chinese family[J].PLoS One,2017,12(5):e0178384.
[12] NIU Z,YAN D,BRESSLER S,et al.A novel splicing mutation in SMPX is linked to nonsyndromic progressive hearing loss[J].International Journal of Pediatric Otorhinolaryngology,2018,104:47-50.
[13] DENG Y,NIU Z,FAN L,et al.A novel mutation in the SMPX gene associated with X-linked nonsyndromic sensorineural hearing loss in a Chinese family[J].Journal of Human Genetics,2018,63(6):723-730.
[14] 施会彬,王玉琴,田志龙,等.伏牛白山羊TACR1基因扩增及生物信息学分析[J].中国畜牧兽医,2018,45(9):2409-2416.SHI H B,WANG Y Q,TIAN Z L,et al.Amplification and bioinformatics analysis of TACR1 gene in Funiu White goat[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2409-2416.(in Chinese)
[15] 罗轶.鸡 FATP1基因cDNA的克隆、组织表达及其生物信息学分析[D].雅安:四川农业大学,2008.LUO Y.cDNA cloning,tissue expression and bioinformatic analysis of fatty acid transport protein 1 in chicken[D].Ya’an:Sichuan Agricultural University,2008.(in Chinese)
[16] 张霞,王淑燕,王配,等.版纳微型猪近交系KITLG基因克隆、组织表达及功能特性分析[J].云南农业大学学报(自然科学),2018,33(2):255-261.ZHANG X,WANG S Y,WANG P,et al.Cloning,tissue expression and functional characteristics of the KITLG gene of Banna Mini-pig inbred line[J].Journal of Yunnan Agricultural University (Natural Science),2018,33(2):255-261.(in Chinese)
[17] 廖珂,柴志欣,钟金城.西藏牦牛HBG基因克隆及生物信息学分析[J].畜牧与兽医,2016,48(3):64-67.LIAO K,CHAI Z X,ZHONG J C.Cloning and bioinformatics analysis on HBG gene in Tibet yak[J].Animal Husbandry & Veterinary Medicine,2016,48(3):64-67.(in Chinese)
[18] 张津闽,张小玉,林森,等.藏系绵羊BMP2基因克隆及组织表达分析[J].基因组学与应用生物学,2018,37(4):1376-1382.ZHANG J M,ZHANG X Y,LIN S,et al.Cloning and tissue expression analysis of BMP2 gene in Tibetan sheep[J].Genomics and Applied Biology,2018,37(4):1376-1382.(in Chinese)
[19] 梁莎莎,庞春英,邓廷贤,等.奶水牛固醇携带蛋白2基因克隆、生物信息学分析及其组织表达检测[J].中国畜牧兽医,2018,45(9):2358-2367.LIANG S S,PANG C Y,DENG T X,et al.Cloning,bioinformatics and tissue expression analysis of SCP2 gene in dairy buffalo[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2358-2367.(in Chinese)
[20] BEATRIZ F,INGRID M P,JUDITH A,et al.The nuclear receptor NOR-1 regulates the small muscle protein,X-linked (SMPX) and myotube differentiation[J].Scientific Reports,2016,6:25944.
[21] LITCHFIELD D W.Protein kinase CK2:Structure,regulation and role in cellular decisions of life and death[J].Biochemical Journal,2003,369(Pt1):1-15.
[22] LARZUL C,LEFAUCHEUR L,ECOLAN P,et al.Phenotypic and genetic parameters for longissimus muscle fiber characteristics in relation to growth,carcass,and meat quality traits in Large White pigs[J].Journal of Animal Science,1997,75:3126-3137.
[23] REN Z Q,XIONG Y Z,DENG C Y,et al.Cloning and identification of porcine SMPX differentially expressed in F1 crossbreds and their parents[J].Acta Biochimica et Biophysica Sinica,2006,38(11):753-758.
[24] BEECKMANN P,SCHR?FFEL J,MOSER G,et al.Linkage and QTL mapping for Sus scrofa chromosome 1[J].Journal of Animal Breeding & Genetics,2015,120(s1):1-10.
[25] DONGIOVANNI P,VALENTI L.Genetics of nonalcoholic fatty liver disease[J].Metabolism Clinical & Experimental,2015,65(8):1026-1037.
[26] 李强子,朱国强,刘吴鑫,等.荷斯坦牛TLR6基因CDS区的生物信息学分析[J].江苏农业学报,2016,32(3):608-614.LI Q Z,ZHU G Q,LIU W X,et al.Bioinformatics analysis of CDS of TLR6 gene in Holstein cattle[J].Jiangsu Journal of Agricultural Sciences,2016,32(3):608-614.(in Chinese)
[27] 营瑞文,张永生,夏欢,等.驴Zfy基因cDNA克隆及生物信息学分析[J].中国畜牧兽医,2018,45(1):39-46.YING R W,ZHANG Y S,XIA H,et al.cDNA cloning and bioinformatics analysis of Zfy gene in Equus asinus[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):39-46.(in Chinese)
[28] SCHINDELER A,LAVULO L,HARVEY R P.Muscle costameric protein,Chisel/Smpx,associates with focal adhesion complexes and modulates cell spreading in vitro via a Rac1/p38 pathway[J].Experimental Cell Research,2005,307(2):367-380.
[29] CHRISTOFFELS V M,HABETS P E,FRANCO D,et al.Chamber formation and morphogenesis in the developing mammalian heart[J].Developmental Biology,2000,223(2):266-278.